Methods for using soy peptides to inhibit h3 acetylation, reduce expression of hmg-coa reductase and increase ldl receptor and sp1 expression in a mammal

ABSTRACT

Controlled studies demonstrate that methods using soy related peptides inhibit H3 acetylation, reduce expression of HMG-CoA reductase and increase LDL receptor and Sp1 expression in mammals. The present disclosure is generally directed to using lunasin peptides and/or lunasin peptide derivatives to 1) inhibit H3 acetylation, 2) reduce expression of HMG-CoA reductase, 3) increase LDL receptor expression or 4) increase Sp1 expression in a mammal. In at least one exemplary embodiment of the present disclosure, an effective amount of lunasin peptides or lunasin peptide derivatives and one or more enzyme inhibitors is provided to a mammal to 1) inhibit H3 acetylation, 2) reduce expression of HMG-CoA reductase, 3) increase LDL receptor expression or 4) increase Sp1 expression in a mammal. Additionally, lunasin will protect against, prevent, or reduce: 1) the expression of Matrix metalloproteinase (MMP-1), 2) collagen breakdown, 3) photoaging and 4) the formation of skin wrinkles.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. patentapplication Ser. No. 11/532,528 filed Sep. 16, 2006. This applicationalso claims the benefit of priority to U.S. patent application Ser. No.12/441,384, filed Mar. 14, 2009, which claims priority to InternationalApplication Number PCT/US07/78584, filed Sep. 15, 2007, which claimspriority to U.S. provisional No. 60/966,529, filed Sep. 16, 2006(formerly U.S. patent application Ser. No. 11/532,526, filed Sep. 16,2006) and claims priority to U.S. provisional No. 61/007,925, filed Jul.17, 2007 (formerly U.S. patent application Ser. No. 11/879,249, filedJul. 17, 2007. All of the above listed applications are herebyincorporated by reference herein in their entireties.

BACKGROUND

1. Technical Field

This disclosure relates generally to a class of peptides that providemammals with a variety of health related benefits. More specifically,the present disclosure related to using soy peptides to inhibit H3acetylation, reduce expression of HMG-CoA reductase, increase LDLreceptor and Sp1 expression, protect against or prevent the expressionof Matrix metalloproteinase (MMP-1), the breakdown of collagen,photoaging and the formation of skin wrinkles.

2. Background Art

Being able to control or manipulate certain important biologicalprocesses provides numerous benefits to researchers and individualsalike. The ability to effect expression of important receptors, enzymesand activators allows researchers to better understand complexbiological mechanisms and create novel and beneficial therapies. Forexample, H3 acetylation, expression of HMG-CoA reductase and LDLreceptor and Sp1 expression in mammals pays a significant role invarious health related factors, including but not limited to total andcholesterol levels, cancer prevention, and UV related skin damage.Accordingly, manipulation and control of these biological mechanisms orfactors would provide numerous health related benefits and provideresearches with new avenues to develop new therapies. Unfortunately,presently there are no known effective methods of safely inhibiting H3acetylation, reducing expression of HMG-CoA reductase and increasing LDLreceptor and Sp1 expression in a mammal. The ability to influence theseand other biological factors would be very beneficial to the fields ofscience and medicine. Accordingly, there exists a need for improvedmethods of inhibiting H3 acetylation, reducing expression of HMG-CoAreductase and increasing LDL receptor and Sp1 expression in a mammal.The present invention provides these and other related benefits.

As used herein, “biological activity” and “bioactivity” refer to the invivo activities of a compound or physiological responses that resultupon in vivo administration of a compound, composition, or othermixture. Biological activity, thus, encompasses therapeutic effects andpharmaceutical activity of such compounds, compositions and mixtures.Biological activities may be observed and measured in in vitro systemsdesigned to test or use such activities also.

As used herein, the term “biologically active” refers to a moleculehaving structural, regulatory and or biochemical functions of anaturally occurring lunasin molecule.

As used herein, a “combination” refers to any association between two oramong more items.

As used herein, the terms “disease” and “disorder” are usedinterchangeably to describe a state, signs, and/or symptoms that areassociated with any impairment of the normal state of a living animal orof any of its organs or tissues that interrupts or modifies theperformance of normal functions, and may be a response to environmentalfactors (such as malnutrition, industrial hazards, or climate), tospecific infective agents (such as worms, bacteria, or viruses), toinherent defect of the organism (such as various genetic anomalies, orto combinations of these and other factors.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., comprising Lunasin) sufficient to effect beneficialor desired results. An effective amount can be administered in one ormore administrations, applications or dosages and is not intended to belimited to a particular formulation or administration route.

As used herein, the terms “administration” and “administering” refer tothe act of giving a drug, pro-drug, or other agent, or therapeutictreatment (e.g., compositions of the present invention) to a subject(e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, andorgans) and/or to direct, instruct, or advise the use of the compositionfor any purpose (preferably, for a purpose described herein). Where theadministration of one or more of the present compositions is directed,instructed or advised, such direction may be that which instructs and/orinforms the user that use of the composition may and/or will provide oneor more of the benefits described herein.

Exemplary routes of administration to the human body can be through theeyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose(nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal,by injection (e.g., intravenously, subcutaneously, intratumorally,intraperitoneally, etc.) and the like.

Administration which is directed may comprise, for example, oraldirection (e.g., through oral instruction from, for example, aphysician, health professional, sales professional or organization,and/or radio or television media (i.e., advertisement) or writtendirection (e.g., through written direction from, for example, aphysician or other health professional (e.g., scripts), salesprofessional or organization (e.g., through, for example, marketingbrochures, pamphlets, or other instructive paraphernalia), written media(e.g., internet, electronic mail, or other computer-related media),and/or packaging associated with the composition (e.g., a label presenton a package containing the composition). As used herein, “written”includes through words, pictures, symbols, and/or other visibledescriptors. Such direction need not utilize the actual words usedherein, but rather use of words, pictures, symbols, and the likeconveying the same or similar meaning are contemplated within the scopeof this invention.

As used herein, the terms “co-administration” and “co-administering”refer to the administration of at least two agent(s) (e.g., compositioncomprising Lunasin and one or more other agents—e.g., a protease enzymeinhibitor) or therapies to a subject. In some embodiments, theco-administration of two or more agents or therapies is concurrent. Inother embodiments, a first agent/therapy is administered prior to asecond agent/therapy. Those of skill in the art understand that theformulations and/or routes of administration of the various agents ortherapies used may vary. The appropriate dosage for co-administrationcan be readily determined by one skilled in the art. In someembodiments, when agents or therapies are co-administered, therespective agents or therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents or therapies lowers the requisite dosage of a potentially harmful(e.g., toxic) agent(s), and/or when co-administration of two or moreagents results in sensitization of a subject to beneficial effects ofone of the agents via co-administration of the other agent.

As used herein, the term “treatment” or grammatical equivalentsencompasses the prevention, improvement and/or reversal of the symptomsof disease (e.g., skin aging). A composition which prevents or causes animprovement in any parameter associated with disease may thereby beidentified as a therapeutic composition. The term “treatment” refers toboth therapeutic treatment and prophylactic or preventative measures.For example, those who may benefit from treatment with compositions andmethods of the present invention include those already with a diseaseand/or disorder (e.g., elevated cholesterol levels) as well as those inwhich a disease and/or disorder is to be prevented (e.g., using aprophylactic treatment of the present invention).

As used herein, the term “at risk for disease” refers to a subject(e.g., a human) that is predisposed to experiencing a particulardisease. This predisposition may be genetic (e.g., a particular genetictendency to experience the disease, such as heritable disorders), or dueto other factors (e.g., age, weight, environmental conditions, exposuresto detrimental compounds present in the environment, etc.). Thus, it isnot intended that the present invention be limited to any particularrisk, nor is it intended that the present invention be limited to anyparticular disease.

As used herein, the terms “individual,” “host,” “subject” and “patient”refer to any animal, including but not limited to, human and non-humananimals (for example, without limitation, primates, dogs, cats, cows,horses, sheep, rodents, poultry, fish, crustaceans, etc.) that isstudied, analyzed, tested, diagnosed or treated. As used herein, theterms “individual,” “host,” “subject” and “patient” are usedinterchangeably, unless indicated otherwise.

As used herein, the term “antibody” (or “antibodies”) refers to anyimmunoglobulin that binds specifically to an antigenic determinant, andspecifically binds to proteins identical or structurally related to theantigenic determinant that stimulated their production. Thus, antibodiescan be useful in assays to detect the antigen that stimulated theirproduction. Monoclonal antibodies are derived from a single clone of Blymphocytes (i.e., B cells), and are generally homogeneous in structureand antigen specificity. Polyclonal antibodies originate from manydifferent clones of antibody-producing cells, and thus are heterogenousin their structure and epitope specificity, but all recognize the sameantigen. Also, it is intended that the term “antibody” encompass anyimmunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from anysource (e.g., humans, rodents, non-human primates, lagomorphs, caprines,bovines, equines, ovines, etc.).

As used herein, the term “antigen” is used in reference to any substancethat is capable of being recognized by an antibody.

As used herein, the terms “Western blot,” “Western immunoblot”“immunoblot” and “Western” refer to the immunological analysis ofprotein(s), polypeptides or peptides that have been immobilized onto amembrane support. The proteins are first resolved by polyacrylamide gelelectrophoresis (i.e., SDS-PAGE) to separate the proteins, followed bytransfer of the protein from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized proteins are thenexposed to an antibody having reactivity towards an antigen of interest.The binding of the antibody (i.e., the primary antibody) is detected byuse of a secondary antibody that specifically binds the primaryantibody. The secondary antibody is typically conjugated to an enzymethat permits visualization of the antigen-antibody complex by theproduction of a colored reaction product or catalyzes a luminescentenzymatic reaction (e.g., the ECL reagent, Amersham).

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. Compounds comprise polypeptidessuch as those described herein.

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a subject, a cell, or a tissue as compared to the same cellor tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., Lunasin) with a carrier, inert oractive, making the composition especially suitable for diagnostic ortherapeutic use in vitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin andmucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells that line hollow organs orbody cavities).

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference).

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the terms “gene expression” and “expression” refer tothe process of converting genetic information encoded in a gene into RNA(e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene(i.e., via the enzymatic action of an RNA polymerase), and for proteinencoding genes, into protein through “translation” of mRNA. Geneexpression can be regulated at many stages in the process.“Up-regulation” or “activation” refer to regulation that increasesand/or enhances the production of gene expression products (e.g., RNA orprotein), while “down-regulation” or “repression” refer to regulationthat decrease production. Molecules (e.g., transcription factors) thatare involved in up-regulation or down-regulation are often called“activators” and “repressors,” respectively.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, transformed celllines, finite cell lines (e.g., non-transformed cells), and any othercell population maintained in vitro.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

As used herein “amino acid” refers to any of the naturally occurringamino acids having the standard designations listed in Table 1, below.It also refers to those known synthetic amino acids. Unless otherwiseindicated, all amino acid sequences listed in this disclosure are listedin the order from the amino terminus to the carboxyl terminus. As usedherein, the abbreviations for any protective groups, amino acids andother compounds, are in accord with their common usage, recognizedabbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature,unless otherwise indicated (see Biochemistry 11: 1726 (1972)). As usedherein, amino acid residues are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

TABLE 1 Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp DGlutamic Acid Glu E Lysine Lys K Arginine Arg R Histidine His H TyrosineTyr Y Cysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser SThreonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu LIsoleucine Ile I Methionine Met M Proline Pro P Phenylalanine Phe FTryptophan Trp W

As used herein, the terms “peptide,” “polypeptide” and “protein” allrefer to a primary sequence of amino acids that are joined by covalent“peptide linkages.” In general, a peptide consists of a few amino acids,typically from 2-50 amino acids. The term “polypeptide” encompassespeptides and proteins, wherein the term “protein” typically refers tolarge polypeptides and the term “peptide” typically refers to shortpolypeptides. In some embodiments, the peptide, polypeptide or proteinis synthetic, while in other embodiments, the peptide, polypeptide orprotein is recombinant or naturally occurring. A “synthetic” peptide isa peptide that is produced by artificial means in vitro (i.e., was notproduced in vivo). The term “peptide” further includes modified aminoacids (whether naturally or non-naturally occurring), such modificationsincluding, but not limited to, phosphorylation, glycosylation,pegylation, lipidization and methylation.

An “isolated peptide” is a peptide which has been substantiallyseparated from components (e.g., DNA, RNA, other proteins and peptides,carbohydrates and lipids) which naturally accompany it in a cell.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80% sequenceidentity, preferably at least 90% sequence identity, more preferably atleast 95% sequence identity or more (e.g., 99% sequence identity).Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

The phrase “functionally equivalent” means that the variant, analogue orfragment of a polypeptide retains a desired biological activity incommon with the lunasin polypeptide. In at least one embodiment of thepresent invention, the desired biological activity in common withlunasin is biological activity related to the control, stabilization, orreduction in production or existing levels of cholesterol, LDLcholesterol, total cholesterol, or lipids. Preferably, a given quantityof the analogue, variant or fragment is at least 10%, preferably atleast 30%, more preferably at least 50, 60, 80, 90, 95 or 99% aseffective as an equivalent amount of the naturally occurring lunasinfrom which the analogue, variant or fragment is derived. Determinationof the relative efficacy of the analogue, variant or fragment canreadily be carried out by utilizing a prescribed amount of the analogue,variant or fragment in the one or more of the assay methods of theinvention and then comparing the ability of the analogue, variant orfragment to naturally occurring lunasin in tests that measure theability of the sample to inhibit the acetylation of histone H3, or toeffect the expression of HMG Co-A reductase, Sp1 or LDL-receptor.

The term “analogue” as used herein with reference to a polypeptide meansa polypeptide which is a derivative of the polypeptide of the invention,which derivative comprises addition, deletion, and/or substitution ofone or more amino acids, such that the polypeptide retains substantiallythe same function as the lunasin polypeptide identified below.

The term “fragment” refers to a polypeptide molecule that is aconstituent of the full-length lunasin polypeptide and possessesqualitative biological activity in common with the full-length lunasinpolypeptide. The fragment may be derived from the full-length lunasinpolypeptide or alternatively may be synthesized by some other means, forexample chemical synthesis. By reference to “fragments” it is intendedto encompass fragments of a protein that are of at least 5, preferablyat least 10, more preferably at least 20 and most preferably at least30, 40 or 50 amino acids in length and which are functionally equivalentto the protein of which they are a fragment.

The term “variant” as used herein refers to a polypeptide which isproduced from a nucleic acid encoding lunasin, but differs from the wildtype lunasin in that it is processed differently such that it has analtered amino acid sequence. For example a variant may be produced by analternative splicing pattern of the primary RNA transcript to that whichproduces wild type lunasin.

Analogues and variants are intended to encompass proteins having aminoacid sequence differing from the protein from which they are derived byvirtue of the addition, deletion or substitution of one or more aminoacids to result in an amino acid sequence that is preferably at least60%, more preferably at least 80%, particularly preferably at least 85,90, 95, 98, 99 or 99.9% identical to the amino acid sequence of theoriginal protein. The analogues or variants specifically includepolymorphic variants and interspecies analogues. The analogues andvariants of the present invention further may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties. One type of conservative amino acid substitutionrefers to the interchangeability of residues having similar side chains.For example, a group of amino acids having aliphatic side chains isglycine, alanine, valine, leucine, and isoleucine; a group of aminoacids having aliphatic-hydroxyl side chains is serine and threonine; agroup of amino acids having amide-containing side chains is asparagineand glutamine; a group of amino acids having aromatic side chains isphenylalanine, tyrosine, and tryptophan; a group of amino acids havingbasic side chains is lysine, arginine, and histidine; and a group ofamino acids having sulfur-containing side chains is cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. More rarely, a variant mayhave “non-conservative” changes (e.g., replacement of a glycine with atryptophan). Similar minor variations may also include amino aciddeletions or insertions (i.e., additions), or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without abolishing biological activity may be foundusing computer programs well known in the art, for example, DNAStarsoftware. Variants can be tested in functional assays such as thosedescribed in the Examples section below.

The term “conservative amino acid substitution” as used herein refers toa substitution or replacement of one amino acid for another amino acidwith similar properties within a polypeptide chain (primary sequence ofa protein). For example, the substitution of the charged amino acidglutamic acid (Glu) for the similarly charged amino acid aspartic acid(Asp) would be a conservative amino acid substitution.

As used herein “lunasin” refers to the natural, synthetically orrecombinantly obtained soybean lunasin polypeptide set forth in (SEQ.ID. 2). Guidance can be found for the identification and screening offunctionally equivalent fragments and analogues of Lunasin peptide inthe following references: U.S. Pat. No. 6,107,287, U.S. Pat. No.6,544,956, US Patent Application 2003/0229038, filed Nov. 22, 2002, U.S.Pat. No. 6,391,848, U.S. patent application Ser. No. 10/252,256, filedSep. 23, 2002, International Application WO 01/72784, filed Mar. 23,2001, and U.S. patent application Ser. No. 10/302,633, filed Nov. 22,2002, all of which are hereby incorporated by reference herein in theirentirety for all purposes. These disclosures will guide one skilled inthe art in identifying functionally equivalent and biologically activefragments, variants and analogues of lunasin.

As used herein “lunasin enriched” refers to compositions containingbiologically active levels of naturally occurring lunasin, or anaturally occurring analogue of lunasin, that is at a concentrationgreater than that at which lunasin is found in the material used as thesource of that lunasin or analogue. As used herein “lunasin enrichedseed extract” refers to compositions containing biologically activelevels of naturally occurring lunasin, or a naturally occurring analogueof lunasin, that is at a concentration at least twice than that at whichlunasin is naturally found in the source seed. Without limiting theinvention to any particular source of the compositions of the presentinvention, lunasin enriched compositions can be obtained from soybean,wheat, barley, soy isolates, soy concentrates, or other soy derivedproducts, whether or not commercially obtained.

As used herein “lunasin protecting soy flour” refers to soy flourcompositions comprising soy flour and an amount of a protease inhibitorsufficient to protect lunasin, or a analogue, variant or fragmentthereof, from complete digestion, wherein the compositions do not havelevels of anti-nutritional elements that would cause an adverse effectin an individual who ingested them.

As used herein “digested” refers to the treatment of a polypeptide witha digestive material that breaks it down into its component amino acids.Examples of digestive materials that can be used are well known in theart, and include, without limitation, pancreatin and other proteasessuch as trypsin, chymotrypsin, pepsin, Proteinase K, thermolysin,thrombin, Arg-C proteinase, Asp-N endopeptidase, AspNendopeptidase+N-terminal Glu, BNPS-Skatole, CNBr, clostripain, formicacid, glutamyl endopeptidase, iodosobenzoic acid, LysC, LysN, NTCB(2-nitro-5-thiocyanobenzoic acid), and Staphylococcal peptidase.

As used herein “partially digested biologically active” in relation to apolypeptide refers to the treatment of a polypeptide with a digestivematerial under conditions that increase the biological activity of thepolypeptide.

The phrase “combination therapy” embraces the administration of acomposition of the present invention in conjunction with anotherpharmaceutical agent that is indicated for treating or preventing adisorder, as part of a specific treatment regimen intended to provide abeneficial effect from the co-action of these therapeutic agents.

Referenced herein are trade names for components including variousingredients utilized in the present invention. The inventors herein donot intend to be limited by materials under a certain trade name.Equivalent materials (e.g., those obtained from a different source undera different name or reference number) to those referenced by trade namemay be substituted and utilized in the descriptions herein.

The compositions herein may comprise, consist essentially of, or consistof any of the elements as described herein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, and protein kinetics, which are within the skill of the art.Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al.,1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait,ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: ALaboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; AnimalCell Culture (R. I. Freshney, ed., 1987); Introduction to Cell andTissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Celland Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths,and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology(Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weirand C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987); PCR: ThePolymerase Chain Reaction, (Mullis et al., eds., 1994); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); and Massisotopomer distribution analysis at eight years: theoretical, analyticand experimental considerations by Hellerstein and Neese (Am J Physiol276 (Endocrinol Metab. 39) E1146-E1162, 1999), all of which areincorporated herein by reference in their entirety. Furthermore,procedures employing commercially available assay kits and reagents willtypically be used according to manufacturer-defined protocols unlessotherwise noted.

SUMMARY OF THE INVENTION

The present invention relates generally to a class of peptides thatprovide mammals with a variety of health related benefits. Morespecifically, the present invention involves using soy peptides toinhibit H3 acetylation, reduce expression of HMG-CoA reductase andincrease LDL receptor and Sp1 expression in a mammal, protect against,prevent, or reduce: 1) the expression of Matrix metalloproteinase(MMP-1), 2) collagen breakdown, 3) photoaging and 4) the formation ofskin wrinkles.

In at least one exemplary embodiment of the present invention, a methodof inhibiting PCAF from acetylating H3 in a mammal is provided. Themethod includes providing an effective amount of lunasin peptides to amammal to inhibit H3 acetylation in the mammal.

In at least one other exemplary embodiment of the present invention, amethod of reducing expression of HMG-CoA reductase in a mammal isprovided. The method includes providing an effective amount of lunasinpeptides to a mammal to reduce expression of HMG-CoA reductase in themammal.

In at least one other exemplary embodiment of the present invention, amethod of increasing LDL receptor expression in a mammal is provided.The method includes providing an effective amount of lunasin peptides toa mammal to increase LDL receptor expression in the mammal.

In at least one other exemplary embodiment of the present invention, amethod of increasing Sp1 transcriptional activator expression in amammal is provided. The method includes providing an effective amount oflunasin peptides to a mammal to increase Sp1 transcriptional activatorexpression in the mammal.

In one aspect of at least one embodiment of the present invention, theeffective amount of lunasin peptides that inhibit H3 acetylation, reduceexpression of HMG-CoA reductase, increase LDL receptor expression orincreases Sp1 transcriptional activator expression in a mammal is 25 to100 mg daily.

In another aspect of at least one embodiment of the present invention,the lunasin peptides include lunasin peptides or lunasin peptidederivatives.

In yet another aspect of at least one embodiment of the presentinvention, the lunasin peptides are obtained from, soy, seed bearingplants other than soy, using recombinant DNA techniques and syntheticpolypeptide production or any combination thereof.

In yet another aspect of at least one embodiment of the presentinvention, the method includes providing an effective amount of one ormore protease enzyme inhibitors to the lunasin peptides.

In at least one exemplary embodiment of the present invention, a methodfor protecting against photoaging of skin in an individual, is provided,comprising: (a) providing: (i) an individual desiring to preventphotoaging of skin and, (ii) a composition comprising a compoundselected from the group consisting of the peptide of SEQ ID NO 2 and afunctionally equivalent variant, fragment or analogue of said peptide;and (b) administering said composition to said subject to protectagainst photoaging.

In one aspect of at least one embodiment of the present invention, saidcompound is obtained from soybean, wheat or barley. In another aspect,the compound is obtained by producing, extracting and purifying saidcompound using recombinant DNA techniques. In yet another aspect, saidcompound is obtained by synthetic polypeptide production. In yet anotheraspect of at least one embodiment of the present invention, saidindividual is a human. In yet another aspect of the present invention,administering comprises topical administration of the composition. Inyet another aspect of the present invention the composition is in theform of a semi-solid formulation, liquid, gel, suspension, or aerosolspray. And in yet another aspect of the invention, said compositionfurther comprises chymotrypsin inhibitor. In still further aspects ofthe present invention, said compound is administered to said individualat between 5 μg/ml and 50 μg/ml.

In at least one exemplary embodiment of the present invention, a methodfor protecting against collagen breakdown in the skin in an individualis provided, comprising: (a) providing: (i) an individual desiring toprevent collagen breakdown in the skin and, (ii) a compositioncomprising a compound selected from the group consisting of the peptideof SEQ ID NO 2 and a functionally equivalent variant, fragment oranalogue of said peptide; and (b) administering said composition to saidsubject to protect against collagen breakdown.

In one aspect of at least one embodiment of the present invention, saidcompound is obtained from soybean, wheat or barley. In another aspect,the compound is obtained by producing, extracting and purifying saidcompound using recombinant DNA techniques. In yet another aspect, saidcompound is obtained by synthetic polypeptide production. In yet anotheraspect of at least one embodiment of the present invention, saidindividual is a human. In yet another aspect of the present invention,administering comprises topical administration of the composition. Inyet another aspect of the present invention the composition is in theform of a semi-solid formulation, liquid, gel, suspension, or aerosolspray. And in yet another aspect of the invention, said compositionfurther comprises chymotrypsin inhibitor. In still further aspects ofthe present invention, said compound is administered to said individualat between 5 μg/ml and 50 μg/ml.

In at least one exemplary embodiment of the present invention, a methodfor protecting against wrinkling of the skin in an individual isprovided, comprising: (a) providing: (i) an individual desiring toprevent wrinkling of skin and, (ii) a composition comprising a compoundselected from the group consisting of the peptide of SEQ ID NO 2 and afunctionally equivalent variant, fragment or analogue of said peptide;and (b) administering said composition to said subject to protectagainst wrinkling of the skin.

In one aspect of at least one embodiment of the present invention, saidcompound is obtained from soybean, wheat or barley. In another aspect,the compound is obtained by producing, extracting and purifying saidcompound using recombinant DNA techniques. In yet another aspect, saidcompound is obtained by synthetic polypeptide production. In yet anotheraspect of at least one embodiment of the present invention, saidindividual is a human. In yet another aspect of the present invention,administering comprises topical administration of the composition. Inyet another aspect of the present invention the composition is in theform of a semi-solid formulation, liquid, gel, suspension, or aerosolspray. And in yet another aspect of the invention, said compositionfurther comprises chymotrypsin inhibitor. In still further aspects ofthe present invention, said compound is administered to said individualat between 5 μg/ml and 50 μg/ml.

BRIEF DESCRIPTION OF DRAWINGS

The above-mentioned features and objects of the present disclosure willbecome more apparent with reference to the following description takenin conjunction with the accompanying drawings wherein like referencenumerals denote like elements and in which:

FIG. 1 shows the 2S albumin protein encoded by Gm2S 1 cDNA (SEQ ID NO1). Arrows indicate endoproteolytic sites that give rise to smallsubunit (“lunasin”) (SEQ ID NO 2) and the large subunit (methionine richprotein). Important regions in both subunits are indicated.

FIG. 2 is a photograph of a Western blot analysis (top) and a table(below) showing densitometer values indicating the relative levels ofexpression of HMG-CoA reductase in HepG2 cells that were (CFM+LS (24))or were not (CFM) treated with lunasin for 24 hours prior to incubationin cholesterol free media (CFM) for 24 hours to activate sterolregulatory element binding proteins (SREBP.) After incubations, totalprotein was extracted and 10 ug protein was loaded onto 10% Tris-glycinegels, electroblotted onto nitrocellulose membrane, and immunostainedwith primary antibodies raised against HMG-CoA reductase and actin (toshow equal loading of proteins.) Spot densitometer values represent meanand standard deviation of data from three separate experiments.

FIG. 3 is a photograph of a Western blot analysis (top) and a table(below) showing densitometer values indicating the relative levels ofexpression of LDL receptor in HepG2 cells that were (CFM+LS(24)) or werenot (CFM) treated with lunasin for 24 hours prior to incubation incholesterol free media (CFM) for 24 hours to activate SREBP. Afterincubations, total protein was extracted and 10 ug protein was loadedonto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane,and immunostained with primary antibodies raised against LDL-receptorand actin (to show equal loading of proteins.) Spot densitometer valuesrepresent mean and standard deviation of data from three separateexperiments.

FIG. 4 is a photograph of a Western blot analysis (top) and a table(below) showing densitometer values indicating the relative levels ofexpression of Sp1 in HepG2 cells that were grown from confluence ingrowth media for 24 hours before growth media was replaced with freshgrowth media (Media), media with lunasin (Media+LS) or cholesterol freemedia with lunasin (CFM+LS) or without lunasin (CFM). Samples were thenincubated for 24 or 48 hours as indicated. After incubations, totalprotein was extracted and 10 ug protein was loaded onto 10% Tris-glycinegels, electroblotted onto nitrocellulose membrane, and immunostainedwith primary antibodies raised against Sp1 and actin (to show equalloading of proteins.) Spot densitometer values represent data from oneexperiment.

FIG. 5 shows the western blots from experiments on PCAF reactionproducts demonstrating that lunasin caused a dramatic reduction inhistone H3 acetylation. Acid extracted protein from untreated (untrt)HeLa cells was used as template in histone acetylase reactions using HATenzyme, PCAF, in the presence or absence of 10 uM lunasin. Reactionproducts were immunoblotted and stained with antibodies againstdiacetylated histone H3. Untrt (−) is the histone template control, NaB(+) correspond to acid extracted histones from NaButyrate treated HeLacells (positive control). Boxed signal indicates significant decrease inH3 acetylation upon addition of 10 uM lunasin compared with no lunasinapplication. Numbers in parenthesis indicate densitometer readingsrelative to the untreated control (set as 1) in PCAF HAT reactionproducts.

FIG. 6 shows the western blots from experiments on PCAF HAT reactionproducts demonstrating that lunasin caused a dramatic reduction inhistone H3 acetylation. Acid extracted histones isolated from untreated(untrt) HeLa cells were used in PCAF HAT reactions, immunoblotted andstained with antibodies to H3 Ac-Lys9 and H3 Ac-Lys14. Untrt (−) is thehistone template control, NaB (+) correspond to acid extracted histonesfrom NaButyrate treated HeLa cells (positive control), +Lun correspondto 10 uM lunasin treated histone template and −Lun correspond tonon-lunasin treated. Boxed signal indicate decreased H3 Lys 14acetylation by PCAF acetylase enzyme in the presence of lunasin. Numbersin parenthesis indicate densitometer readings relative to the signallevel of lunasin/lunasin treatment (set as 1) in immunoblots stainedwith Ac-Lys14H3.

DETAILED DESCRIPTION

Lunasin is a recently discovered bioactive component in soy with a novelchromatin-binding property and epigenetic effects on gene expression (1,2). The lunasin soy peptide is heat stable, water soluble and found insignificant amounts in select soy protein preparations (3). Studies showthat it can get inside mammalian epithelial cells through its RGD celladhesion motif, bind preferentially to deacetylated histones and inhibithistone H3 and H4 acetylation (4). There is growing evidence thatcellular transformation, responses to hormones and dietary andenvironmental effects involve epigenetic changes in gene expression,which are modulated by the reversible processes of DNAmethylation-demethylation and histone acetylation-deacetylation (5, 6).Lunasin is the first natural substance to be identified as a histoneacetylase inhibitor, although it does not directly affect the histoneacetylase enzyme. It inhibits H3 and H4 acetylation by binding tospecific deacetylated lysine residues in the N-terminal tail of histonesH3 and H4, making them unavailable as substrates for histoneacetylation. The elucidation of the mechanism of action makes lunasin animportant molecule for research studies to understand the emerging roleof epigenetics and chromatin modifications in important biologicalprocesses.

The study on the effect of lunasin on prostate carcinogenesis at theUniversity of California at Davis revealed the effects of lunasin onhistone H4 modifications and the up regulation of chemopreventive genes,(7). However, until now, the specific effect of lunasin binding todeacetylated H3N-terminal tail and the inhibition of H3 histoneacetylation in biological systems had not yet been investigated. Todetermine the specific biological effect of lunasin binding todeacetylated histone H3 and inhibition of acetylation, the induction ofgenes involved in cholesterol biosynthesis by the sterol regulatoryelement binding proteins (SREBP) was chosen as a biological model. Thisbiological model was chosen because activation of SREBPs by steroldepletion results in the increased acetylation of histone H3 but nothistone H4, by the histone acetylase enzyme PCAF, in chromatin proximalto the promoters of HMG-CoA reductase and the LDL receptor genes (8) andSREBP activation results in the increased recruitment of co-regulatoryfactors, CREB to the promoter of HMG-CoA reductase gene, and Sp1 to thepromoter of LDL receptor gene (8).

Our studies on in vitro histone acetylase (HAT) assays show that lunasinsignificantly inhibits histone H3 acetylation (specifically lysine 14 inH3N-terminal tail) by the histone acetylase enzyme, PCAF. Cell cultureexperiments using HepG2 liver cells show that synthetic lunasin cansignificantly reduce HMG-CoA reductase expression and increase LDLreceptor gene expression in cholesterol-free media similar to theeffects of statin (cholesterol-lowering) drugs. Our studies have alsoshown that the increase in LDL receptor expression coincides with theincrease in Sp1 expression in cholesterol-free media. Based on thesestudies, a molecular mechanism of action is proposed wherein syntheticlunasin reduces total and LDL cholesterol levels by binding todeactylated histone H3 and inhibiting histone H3 acetylation by PCAF(through its association with the CREB-binding protein), therebyreducing SREBP activation of the HMG-CoA reductase gene resulting inlower endogenous cholesterol biosynthesis, and by increasing theexpression of the Sp1 co-activator in sterol-free media and upon SREBPactivation, an increased amount of membrane bound LDL receptors isexpressed leading to significant reduction of plasma LDL cholesterollevels (9).

Our data described and shown below demonstrates that lunasin (a.k.a.lunastantin) is the bioactive agent from soy responsible for inhibitingH3 acetylation, reducing expression of HMG-CoA reductase and increasingLDL receptor and Sp1 expression in a mammal.

Our surprising finding that lunasin inhibits H3 acetylation, reducesexpression of HMG-CoA reductase and increases LDL receptor and Sp1expression in a mammal can be used for numerous health related benefits,including but not limited to, to lower total or LDL cholesterol levelsor to prevent, control or treat cancers in mammals. These effects oflunasin can be further increased by developing formulations of lunasinand lunasin derivatives that are optimized for adsorption and deliveryto the liver.

Lunasin is the small subunit peptide of a cotyledon-specific 2S albumin.FIG. 1 shows the 2S albumin protein and the small lunasin subunit. Ithas been shown that constitutive expression of the lunasin gene inmammalian cells disturbs kinetochore formation and disrupts mitosis,leading to cell death (2). When applied exogenously in mammalian cellculture, the lunasin peptide suppresses transformation of normal cellsto cancerous foci that are induced by chemical carcinogens andoncogenes. To elucidate its chemopreventive mechanism of action, we haveshown that lunasin (a) is internalized through its RGD cell adhesionmotif, (b) colocalizes with hypoacetylated chromatin in telomeres atprometaphase, (c) binds preferentially to deacetylated histone H4, whichis facilitated by the presence of a structurally conserved helical motiffound in other chromatin-binding proteins, (d) inhibits histone H3 andH4 acetylation, and (e) induces apoptosis in E1A-transfected cells (4).Based on these results, a novel chemopreventive mechanism has beenproposed wherein lunasin gets inside the nucleus, binds to deacetylatedhistones, prevents their acetylation and inhibits gene expression likethose controlled by the Rb tumor suppressor and h-ras oncogene.

Lunasin's Effect on Expression of Sp1 Coactivator

Inhibition of H3 histone acetylation by PCAF histone acetylase enzyme isrequired for the SREBP activation of genes involved in cholesterolbiosynthesis including HMG-CoA reductase (9). Previous study has shownthat lunasin is a potent inhibitor of histone H3 acetylation inmammalian cells exposed to the histone deacetylase inhibitor, sodiumbutyrate (NaB) (4). To determine the effect of lunasin on histone H3acetylation by PCAF, HAT assay reaction using acid-extracted histonesfrom untreated HeLa cells as template was conducted. Immunoblottedreaction products have been stained with antibodies against diacetylatedhistone H3 (Ac-Lys9+Ac-Lys14) and the details of our experiment and itsresults are shown and described in FIG. 5. In brief, the HAT enzyme,PCAF, is shown to increase significantly histone H3 acetylation in theabsence of lunasin (35-fold increase). However, the addition of lunasinin the PCAF reaction, resulted in dramatic reduction of histone H3acetylation, indicating that lunasin is a potent inhibitor of histone H3acetylation catalyzed by the PCAF acetylase enzyme.

To determine the specific lysine residue in histone H3 that is inhibitedby lunasin from being acetylated, immunoblotted products of PCAFacetylase reactions were hybridized with antibodies raised againstacetylated Lys 9 and acetylated Lys 14 in H3 terminal tails. The resultsand details of our experiments, as shown and described in FIG. 6,demonstrate that lunasin specifically binds to Lys 14, preventing itfrom being acetylated by PCAF.

In one exemplary embodiment of the present invention, a method ofinhibiting H3 acetylation in a mammal is provided. The method includesproviding an effective amount of lunasin peptides to a mammal to inhibitH3 acetylation in the mammal.

In another exemplary embodiment of the present invention, a method ofreducing expression of HMG-CoA reductase in a mammal is provided. Themethod includes providing an effective amount of lunasin peptides to amammal to reduce expression of HMG-CoA reductase in the mammal.

In yet another exemplary embodiment of the present invention, a methodof increasing LDL receptor expression in a mammal is provided. Themethod includes providing an effective amount of lunasin peptides to amammal to increase LDL receptor expression in the mammal.

In yet another exemplary embodiment of the present invention, a methodof increasing Sp1 transcriptional activator expression in a mammal isprovided. The method includes providing an effective amount of lunasinpeptides to a mammal to increase Sp1 transcriptional activatorexpression in the mammal.

In one aspect of at least one embodiment of the present invention, theeffective amount of lunasin peptides that inhibit H3 acetylation, reduceexpression of HMG-CoA reductase, increase LDL receptor expression orincreases Sp1 transcriptional activator expression in a mammal is 25 to100 mgs daily. It should be appreciated that the effective amount oflunasin will depend, at least in part, on the size, weight, health anddesired goals of the mammals consuming the compositions. Accordingly, itis believed that in at least one embodiment, the effective amount oflunasin provided to the mammal is 25 mg to 100 mg daily.

In another aspect of at least one embodiment of the present invention,the lunasin peptides include lunasin peptides or lunasin peptidederivatives. It should also be appreciated that the present inventionincludes the use of lunasin peptide derivatives, which are any peptidesthat contain the same functional units as lunasin. It should also beappreciated the products and compositions of the present invention canbe used in, foods, powders, bars, capsules, shakes and other well knownproducts consumed by mammals or used separately.

In yet another aspect of at least one embodiment of the presentinvention, the lunasin peptides are obtained from, soy, seed bearingplants other than soy, using recombinant DNA techniques and syntheticpolypeptide production or any combination thereof.

In yet another aspect of at least one embodiment of the presentinvention, the method includes providing an effective amount of one ormore protease enzyme inhibitors with or without the lunasin peptides.The protease enzyme inhibitors act to protect lunasin from digestion andfacilitate absorption and delivery to the appropriate target areas.Examples of appropriate protease enzyme inhibitors include, but are notlimited to, pancreatin, trypsin and/or chymotrypsin inhibitors. Itshould be appreciated that the scope of the present inventions includesthe use of the lunasin and/or lunasin derivatives with any othercomposition or product that is known or believed to facilitate lunasin'sabsorption or delivery in a mammal.

Photoprotective Effects.

The activity of lunasin peptides in prevention of histone H3 acetylationhas additional applications for the protection against and prevention ofthe expression of Matrix metalloproteinase (MMP-1), collagen breakdown,photoaging and the formation of skin wrinkles.

The present invention encompasses the use of lunasin peptides for theprotection against and prevention of the expression of Matrixmetalloproteinase (MMP-1), collagen breakdown in the skin, photoaging ofthe skin, premature aging of the skin and the formation of skinwrinkles.

It has been previously shown that UV irradiation stimulates histone H3acetylation at Lys14 (19). Further, an increase in histone H3acetylation results in chromatin modification of gene promoters and isassociated with increased accessibility of chromatin by transcriptionalfactors involved in the activation and expression of repressed genes(19). The histone acetylase (HAT) enzyme that specifically acetylatesH3-Lys14 is PCAF (p300/CBP-associated factor) is the mammalian homologueof the yeast HAT, Gcn5p that was evaluated in the studies referenced.

Matrix metalloproteinase (MMP-1), commonly known as interstitialcollagenase, is known to degrade collagen and extracellular matrixcomponents (20). Its expression promotes the breakdown of collagen andconnective tissues, and excessive damage to connective tissue isassociated with dermal photoaging (20). MMP-1 triggers long termdetrimental effects such as premature aging. UV radiation from the suninduces the expression of MMP-1 by increasing histone H3 acetylation(20). Inhibition of histone H3 acetylation by HAT inhibitors reducesUV-induced expression of MMP-1 (20). It is thus desirable to interferewith the acetylation of histone H3 in order to reduce the expression ofMMP-1 and as a result reduce collagen breakdown and the photoaging andthe premature aging associated with it.

As shown herein, the binding of lunasin to H3 masks deacetylatedH3-Lys14 from acetylation, and, as a result lunasin inhibits H3acetylation by PCAF.

Without being limited to any particular mechanism of action, it isbelieved that by binding to chromatin, specifically to the N-terminaltail of deacetylated histone H3, lunasin will prevent the UV-inducedPCAF acetylation of H3-Lys14 in the chromatin near the MMP-1 promoter,thereby interfering with the expression of MMP-1 and preventing thedamage to collagen, photoaging, premature skin aging and skin wrinkling.The inhibition of H3 acetylation in the epidermal layer of skin cellswill interfere with the expression of MMP-1 that would otherwise beactivated by exposure to UV radiation from the sun. The interferencewith MMP-1 expression as a result of lunasin treatment will lead to adecrease in the expression of MMP-1, will protect against collagenbreakdown in the skin and result in protection against the effects ofphotoaging and premature aging of the skin and protection against skinwrinkling that are the result of sun exposure.

Administration

The compositions can be administered using a number of different routesincluding oral administration, topical administration, transdermaladministration, or injection directly into the body. Administration ofcompositions for use in the practice of the present invention can besystemic (i.e., administered to the subject as a whole via any of theabove routes) or localized (i.e., administered to the specific locationof the particular disease or pathological condition of the subject viaany of the above routes). In a preferred embodiment of the presentinvention, the compositions to decrease the expression of Matrixmetalloproteinase (MMP-1), protect against collagen breakdown in theskin, protect against skin photoaging, or protect against skin wrinklingin an individual are administered by topical administration.

The present methods, kits, and compositions can also be used in“combination therapy” with another composition or treatment that isindicated for treating or preventing a disorder.

Dosing

In one exemplary embodiment of the present invention, a productcontaining an effective amount of lunasin peptides to prevent theexpression of Matrix metalloproteinase (MMP-1), reduce collagenbreakdown, or prevent dermal photoaging in an individual that is treatedwith the product is provided.

Depending upon the particular needs of the individual subject involved,the compositions of the present invention can be administered in variousdoses to provide effective treatment concentrations based upon theteachings of the present invention. Factors such as the activity of theselected compositions, the physiological characteristics of the subject,the extent or nature of the subject's disease or pathological condition,and the method of administration will determine what constitutes aneffective amount of the selected compositions. Generally, initial doseswill be modified to determine the optimum dosage for treatment of theparticular subject. Suitable dosages can be chosen by taking intoaccount any or all of such factors as the size, weight, health, age, andsex of the human or individual, the desired goals of the patient, theseverity of the pathological condition for which the composition isbeing administered, the response to treatment, the type and quantity ofother medications being given to the patient that might interact withthe composition, either potentiating it or inhibiting it, and otherpharmacokinetic considerations such as liver and kidney function. Theseconsiderations are well known in the art and are described in standardtextbooks.

A therapeutically effective amount of any embodiment of the presentinvention is determined using methods known to pharmacologists andclinicians having ordinary skill in the art. Blood levels of thecomposition can be determined using routine biological and chemicalassays and these blood levels can be matched to the route ofadministration. The blood level and route of administration giving themost desirable level of cholesterol reduction can then be used toestablish an “effective amount” of the pharmaceutical composition fortreatment.

This same method of titrating a composition in parallel withadministration route can be used to ascertain a therapeuticallyeffective amount of the compositions of the present invention fortreating any and all disorders described herein. In addition, animalmodels as described below can be used to determine applicable dosages totreat or prevent a particular disease or pathological condition.Typically, dosage-effect relationships from in vitro or in vivo testsinitially can provide useful guidance on the proper doses for subjectadministration.

In one embodiment of the present invention related to decreasing theexpression of Matrix metalloproteinase (MMP-1), protecting againstcollagen breakdown in the skin, protecting against skin photoaging, orprotecting against skin wrinkling in an individual, methods andcompositions of the invention encompass a dose of a compositioncomprising lunasin, or a functionally equivalent variant, analogue orfragment of lunasin, of about 5 ng to about 1000 g, or about 100 ng toabout 600 mg, or about 1 μg to about 500 μg, or about 5 μg/ml and 50μg/ml. Illustratively, a dosage unit of a composition of the presentinvention can typically contain, for example, without limitation, about5 ng, 50 ng 100 ng, 500 ng, 1 μg, 10 μg, 100 μg, 250 μg, 500 μg, 1 mg,10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 700 mg, 800 mg, 900mg, 1 g, 5 g, 10 g, 20 g, 30 g, or 40 g of a composition of the presentinvention. In certain preferred embodiments of the present invention,compositions of the present invention contain about 1 to 500 μg,preferably 5 μg/ml and 50 μg/ml, more preferably approximately 250 μgper dosage of lunasin, or fragments, variants and analogues of lunasin.

Exemplary dosages for lunasin, or fragments, variants and analoguesthereof, in accordance with the teachings of the present invention,range from 0.1 μg to 200 mg, preferably, 1 μg to 100 mg, more preferably25 μg to 500 μg for humans and other individuals, although alternativedosages are contemplated as being within the scope of the presentinvention.

In certain preferred embodiments of the present invention forcompositions and methods for topical administration, lunasin, orfragments, variants and analogues thereof, is present at a level ofbetween 25 μg/ml and 25 mg/ml, more preferably between 50 μg/ml and 1mg/ml, more preferably between 100 μg/ml and 500 μg/ml, even morepreferably, approximately 250 μg/ml.

Existing literature offers additional guidance in determiningappropriate dosage for topical administration of lunasin or fragments,variants and analogues thereof for applications of the presentinvention. Guidance on a lunasin effective dosage range can be obtained,for example, from Table 1 of reference (4) below (Galvez, A F, et al.,Cancer Res. 61:7473-7478 (2001)), wherein dosages of 10 nM to 10,000 nMof lunasin were evaluated to determine lunasin efficacy in reducingtumor formation in mammalian cells. Lunasin dosages of 1,000 nM(equivalent to 5 μg/ml) to 10,000 nM (equivalent to 50 μg/ml) all showedstatistically significant tumor reduction activity compared to thecontrol group, but were not statistically significantly different intheir ability to reduce tumor formation induced by chemical carcinogens.The chemopreventive effect of lunasin is linked to the ability oflunasin to bind to deacetylated histones and inhibit histone acetylation(4). This and other dosage guidance in the literature will be helpful indetermining appropriate dosage for various topical applicationsincluding those related to decreasing the expression of Matrixmetalloproteinase (MMP-1), protecting against collagen breakdown in theskin, protecting against skin photoaging, or protecting against skinwrinkling in an individual.

In certain preferred embodiments of the present invention forcompositions and methods for oral administration, lunasin, or fragments,variants and analogues thereof, is provided to an individual at a levelof between 0.01 mg/Kg and 100 mg/Kg body weight of an individual,preferably 0.05 mg/Kg and 50 mg/Kg, more preferably between 0.5 mg/Kgand 2.5 mg/Kg, and even more preferably between 0.2 mg/Kg and 1.5 mg/Kg.

A dose can be administered in one to about four doses per day, or in asmany doses per day to elicit a therapeutic effect. The dosage form canbe selected to accommodate the desired frequency of administration usedto achieve the specified dosage, as well as the route of delivery.

The amount of therapeutic agent necessary to elicit a therapeutic effectcan be experimentally determined based on, for example, the absorptionrate of the agent into the blood serum, or the dermal layer of the skinfor topical applications, and the bioavailability of the agent.Determination of these parameters is well within the skill of the art.

Formulations.

The invention also concerns formulations containing the compositions ofthe present invention. The products and compositions of the presentinvention can be used alone or in foods, powders, bars, capsules, shakesand other well known products consumed by individuals.

In one preferred embodiment the compositions of the present inventionare together with a dietary suitable excipient, diluent, carrier, orwith a food. In a preferred embodiment of the present invention, theformulation is in the form of a pill, tablet, capsule, powder, food baror similar dosage form.

The formulations may be a variety of kinds, such as nutritionalsupplements, pharmaceutical preparations, vitamin supplements, foodadditives or foods supplemented with the specified compositions of theinvention, liquid or solid preparations, including drinks, sterileinjectable solutions, tablets, coated tablets, capsules, powders, drops,suspensions, or syrups, ointments, lotions, creams pastes, gels, or thelike.

The formulations may be packaged in convenient dosage forms, and mayalso include other active ingredients, and/or may contain conventionalexcipients, pharmaceutically acceptable carriers and diluents. Theinclusion of the compositions of the present invention in herbalremedies and treatments is also a preferred part of the invention.

Preferred formulations for topical applications of the compositions ofthe present invention for both pharmaceutical and cosmetic use willemploy excipients that are suitable for topical application. Topicalformulations typically are gels, salves, powders, or liquids, thoughcontrolled formulations which release defined amounts of activeingredient at the desired surface are also desirable. The formulationsmay contain materials which enhance the permeability of the activemoieties through the epidermis. Such penetrants include, for example,DMSO, various bile salts, non-toxic surfactants and the like. Standardingredients for cosmetic/pharmaceutical compositions are well known inthe art; formulations for topical application of pharmaceuticals arefound in Remington's Pharmaceutical Sciences, latest edition, MackPublishing Co., Easton, Pa., incorporated herein by reference. Cosmeticformulations are widely varied and well known to practitioners.

While the products, compositions and related methods have been describedin terms of what are presently considered to be the most practical andpreferred embodiments, it is to be understood that the disclosure neednot be limited to the disclosed embodiments. It is intended to covervarious modifications and similar arrangements included within thespirit and scope of the claims, the scope of which should be accordedthe broadest interpretation so as to encompass all such modificationsand similar structures. The present disclosure includes any and allembodiments of the following claims. All the patents, journal articlesand other documents discussed or cited above are herein incorporated byreference.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Lunasin Reduces Expression of HMG-CoA Reductase, IncreasesExpression of LDL Receptor

The lowering of serum cholesterol by statin drugs is achieved bycompetitively inhibiting the HMG-CoA reductase, the rate limiting enzymein the body's metabolic pathway for synthesis of cholesterol. Byreducing endogenous cholesterol synthesis, statins also cause livercells to up regulate expression of the LDL receptor, leading toincreased clearance of low-density lipoprotein (LDL) from thebloodstream (9). In 1985, Michael Brown and Joseph Goldstein receivedthe Nobel Prize in Medicine for their work in clarifying thisLDL-lowering mechanism.

Transcriptional regulation of HMG-CoA reductase and LDL receptor iscontrolled by the Sterol Regulatory Element-Binding Protein-1 and -2(SREBP). This protein binds to the sterol regulatory element (SRE)located on the 5′ end of the reductase and the LDL receptor genes. WhenSREBP is inactive, it is bound to the ER or nuclear membrane. Whencholesterol levels fall, SREBP is released from the membrane byproteolysis and migrates to the nucleus, where it binds to the SRE to upregulate transcription of HMG-CoA reductase and LDL receptor (8, 9).

In cell culture of HepG2 liver cells, it is possible to activate SREBPand increase the expression of HMG-CoA reductase and LDL-receptor byremoving cholesterol in the growth media. This can be achieved byexposing the cells to serum-free media for 24 hours (15, 16).

The following related experiments were performed to evaluate the effectof lunasin on HMG-CoA reductase expression and LDL-receptor expression.

In the first experiment, HepG2 cells (1×10⁶) were treated with orwithout 10 uM synthetic lunasin in DMEM with 10% FBS for 24 hours beforegrowth media was replaced with cholesterol-free media to activate SREBP.After 24 hours, total protein was extracted and 10 ug protein was loadedonto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane,and immunostained with primary antibodies raised against HMG-CoAreductase and actin (to show equal loading of proteins). Spotdensitometer values are obtained by digital scanning and Un-Scan Itsoftware, and represent mean and standard deviation of data from threeseparate experiments. The results are shown in FIG. 2.

In the second experiment, HepG2 cells (1×10⁶) were treated with orwithout 10 uM synthetic lunasin in DMEM with 10% FBS for 24 hours beforegrowth media is replaced with cholesterol-free media to activate SREBP.After 24 hours, total protein was extracted and 10 ug proteins loadedonto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane,and immunostained with primary antibodies raised against LDL-receptorand actin (to show equal loading of proteins). Spot densitometer valueswere obtained by digital scanning and Un-Scan It software, and representmean and standard deviation of data from three separate experiments. Theresults are shown in FIG. 3.

FIGS. 2 and 3 show the upregulation of HMG-CoA reductase (98% increase)and LDL-receptor (34% increase) when HepG2 cells are grown incholesterol-free media for 24 hours. However when lunasin is added tothe cholesterol-free media, the expression of the HMG-CoA reductase isreduced by more than 50% (FIG. 2), while the expression of LDL-receptorhas increased by more than 60% (FIG. 3).

This effect of lunasin is similar to statin drugs that reducesendogenous cholesterol synthesis by inhibiting HMG-CoA reductaseactivity, which leads to increased LDL receptor expression. However,while it is not intended that the present invention be limited to anyprecise mechanism or mode of action, the mode of action of lunasin isbelieved to differ from statin drugs in that it appears to inhibitexpression of HMG-CoA reductase at the transcriptional level, ratherthan on inhibiting its enzyme activity. Like statin drugs, lunasin upregulates the expression of LDL-receptor gene. Again, while it is notintended that the present invention be limited to any precise mechanismor mode of action, the contrasting effect of lunasin on these twoSREBP-controlled genes can be explained by the selective recruitment ofdifferent co-regulatory transcription factors to two separatecholesterol-regulated promoter/regulatory sequences.

Example 2 Lunasin's Effect on Expression of Sp1 Coactivator

Unlike HMG-CoA reductase, SREBP activation of LDL-receptor by steroldepletion requires increased recruitment of Sp1 co-activator to a siteadjacent to SREBP in the promoter/regulatory sequence of LDL-receptorgene (25). As shown in FIG. 3, the up regulation of LDL-receptor bylunasin (LS) in cholesterol-free media may be due to increasedavailability and recruitment of the Sp1 coactivator to the LDL-receptorpromoter/regulatory sequence. To test this hypothesis, the level of Sp1was determined in lunasin-treated growth media and cholesterol-freemedia by Western analysis using Sp1 antibody, as follows: HepG2 cells(1×10⁶) were grown from confluence in DMEM with 10% FBS for 24 hoursbefore growth media was replaced with fresh growth media orcholesterol-free media (to activate SREBP) and treated with, or without10 uM synthetic lunasin. After 24 hours, total protein was extractedfrom each treatment and 10 ug protein loaded onto 10% Tris-glycine gels,electroblotted onto nitrocellulose membrane, and immunostained withprimary antibodies raised against Sp1 and actin (to show equal loadingof proteins). Spot densitometer values were obtained by digital scanningand Un-Scan It software and represent data from one experiment. Theresults are shown in FIG. 4.

FIG. 4 shows that Sp1 levels in control and lunasin-treated growth mediawere not significantly different. However, Sp1 levels increased incholesterol-free media by 23%, compared to the growth media. Theaddition of lunasin in the cholesterol-free media further increased Sp1levels by almost 60%, which closely mirrors the increase in LDL-receptorlevels in lunasin-treated, cholesterol-free media.

The data from these experiments indicate that the increase inLDL-receptor expression by lunasin in sterol-depleted media could beattributed to the increased availability of the Sp1 transcriptionalco-activator. Also, the inhibition of HMG-CoA reductase expression bylunasin lowers intracellular cholesterol levels that keep SREBPactivated, resulting in the upregulation of LDL receptor expression.

Therefore, the data shows that lunasin inhibits the expression ofHMG-CoA reductase, the rate limiting enzyme in the body's metabolicpathway for synthesis of cholesterol, and at the same time increases theexpression of the LDL receptor, leading to increased clearance oflow-density lipoprotein (LDL) from the bloodstream, which will lowertotal and LDL cholesterol in a mammal.

Most circulating cholesterol in mammals is synthesized internally, onaverage 1000 mg/day compared to 200-300 mg/day from intestinal intake ina human diet. Thus the internal production of cholesterol, as catalyzedby HMG-CoA reductase and the amount of LDL receptors in liver cellmembranes, is the single most important factor in modulating cholesterollevels in mammals. Accordingly, these experiments demonstrate that aneffective amount of lunasin reduces both LDL and total cholesterollevels in a mammal.

Example 3

Lunasin can be extracted from commercial sources of soy protein. Lunasinhas been found in significant amounts from commercial sources of soyprotein and its homologues from other seed sources such as barley andwheat. To identify preferred sources for the starting raw material thatcan be used for lunasin extraction, several commercially available soyprotein products were screened for the presence of lunasin.

The procedure used was as follows: approximately 500 mg of soy proteinsamples obtained from different commercial sources (Solae, St. Louis,Mo.) were dissolved in 50 mL of aqueous phosphate buffer (pH 7.2) byshaking for 1 hour at room temperature. Samples were centrifuged at 2500rpm for 30 minutes and the aqueous fraction separated and put inseparate tubes. Protein concentrations were measured by Bradford assayand around 20 ug of total protein were loaded onto two Bio-RadLaboratories (Hercules, Calif.) 16% Tris-Tricine gels. One of theSDS-PAGE gels (I) was stained with Coomasie blue and destained beforedigital imaging. The 5 kDa lunasin band is indicated by arrow. The other(II) is electroblotted onto nitrocellulose membrane and incubated withaffinity-purified lunasin polyclonal antibody (Pacific Immunology,Ramona, Calif.) followed by HRP-conjugated donkey anti-rabbit secondaryantibody (Amersham Biosciences, Piscataway, N.J.). Lunasin immunosignals(indicated by arrow) are detected using the ECL Western blotting kitfrom Amersham.

The results showed that lunasin concentration varies dramatically fromsource to source. This assay is a useful tool in identifying sources ofnatural lunasin for use in the compositions and methods of the presentinvention. The soy concentrate that contained the most lunasin was usedas a starting material in a buffer extraction procedure to produce thelunasin-enriched soy concentrate (LeSC) used in the followingexperiments.

Example 4

Formulated lunasin-enriched soy concentrate (LeSC) and LeSC supplementedwith soy flour (SF) contain significant amounts of lunasin. Thisexperiment evaluated the amount of lunasin in lunasin-enriched soyconcentrate (LeSC) and LeSC supplemented with soy flour.

Lunasin-enriched soy concentrate was produced by first identifyingcommercially available soy protein preparations that contain significantamounts of lunasin by Western blot analysis using lunasin polyclonalantibody, as described in Example 3. The soy protein concentrateidentified to contain the most lunasin was used as starting material ina one-step buffer extraction procedure (0.1×PBS pH 7.2) followed bycentrifugation to separate the supernatant. Two volumes of acetone wereadded to supernatant and precipitate was separated by centrifugationwith filter bags before vacuum drying to get the lunasin-enriched soyconcentrate.

Efforts to make lunasin more resistant to undesired excessive digestion,improve its bioavailability, and retain its bioactivity when ingested,resulted in the discovery of at least one of the preferred embodimentsof the present invention, a composition comprising lunasin enriched soyconcentrate and soy flour.

In at least one embodiment of the present invention, compositions of thepresent invention that comprise naturally derived lunasin can beoptimized for use in particular methods of the present invention byvarying the amount of total protein and lunasin content, which can becontrolled by the amount of soy concentrate used, and varying the amountof lunasin protection from digestion, which can be controlled by theamount of minimally heated soy flour used.

For food based items it is sometimes desirable to limit the amount ofprotease inhibitors in a product. For example, U.S. Patent ApplicationNo. 20070092633, filed Apr. 26, 2007, hereby incorporated by reference,teaches that part of the standard processing of some soy productsincludes heat treatment to inactivate anti-nutritional elements such asBowman-Birk and Kuntz inhibitors. Therefore, in a preferred embodimentof the present invention, a composition comprising lunasin and soy flouris optimized through preparation methods describe herein or known to oneskilled in the art, to have a level of protease inhibitors sufficient toprotect lunasin biological activity during digestion but not sufficientto have levels of anti-nutritional elements that are undesirable fororal use.

Clinical trials on a 50:50 blend of soy concentrate and soy flour led toa 20-30% reduction of LDL cholesterol (17, 18.) Those clinical trialswere performed without the knowledge that lunasin is an active elementin soy concentrate in reducing LDL cholesterol, and therefore did notcontrol for the level of lunasin present in the blend. The presentinvention teaches improved methods of determining lunasin concentrationin starting materials and final products of the present invention, so asto maximize the concentration of lunasin and therefore the activity ofcompositions of treatment in cholesterol related applications. In atleast one preferred embodiment of the present invention the ratio of soyflour to soy concentrate is between 10:90 and 50:50, more preferablybetween 20:80 and 40:60, more preferably approximately 30:70 soyflour:soy concentrate. This ratio for minimally heated soy flour and soyconcentrate was determined to provide a biologically activeconcentration of lunasin and as well as sufficient protection fromdigestion by the soy flour.

In the following several experiments, minimally heated soy flour (SF)was added to the starting soy concentrate (at a 30:70 w/w mixture)before buffer extraction with 0.1×PBS pH 7.2 and acetone precipitationto produce lunasin enriched soy concentrate plus soy flour (LeSC+SF.)

The Western blotting analysis procedure used in this experiment was asfollows: approximately 20 ug of total protein from LeSC, SF and theLeSC+SF were electrophoresed in 16% Tris-Tricine gels and electroblottedonto nitrocellulose membrane. Blots were incubated with lunasinpolyclonal antibody followed by HRP-conjugated anti-rabbit secondaryantibody before lunasin immunosignals were detected with the ECL kit.The results showed that both LeSC and LeSC+SF contained significantamounts of lunasin.

Example 5

Lunasin-enriched soy concentrate with soy flour (LeSC+SF) retainsbioactivity even when digested with digestive enzymes. Biologicalactivity of LeSC (A), LeSC+SF (B), digested LeSC+SF (C), digested LeSC(D), digested soy protein isolate (E) and digested soy concentrate (F)was measured using the H3 histone acetyltransferase (HAT) assay (seeExample 8.) Around 100 mg total protein of LeSC, LeSC+SF, soy proteinisolate and soy concentrate were digested by mixing pancreatin (SigmaLife Sciences, Saint Louis, Mo.) at 1:1 (w/w) and incubating for 30 min.at 40° C. To confirm that the HAT assay is working, treatment withsynthetic lunasin (+synL) was included. Synthetic lunasin reducedacetylation of histone H3 by the histone acetylase enzyme, PCAF, usingcore histones isolated from chicken erythrocyte (Upstate/Millipore,Billerica, Mass.) as template for the HAT assay. Around 10 ug of sampleprotein was incubated with 1 ug of core histones before undergoing HATreaction with PCAF enzyme and acetyl CoA substrate. Reaction productswere run on 16% Tris-Tricine gels and electroblotted onto nitrocellulosemembrane. Blots were incubated with primary antibody raised againstacetylated H3 (diacetylated at histone14 and histone10) andHRP-conjugated anti-rabbit secondary antibody before detecting signalsusing the ECL kit. Low signals indicated that the lunasin peptide wasbioactive because it prevented the acetylation of histone H3. Strongsignals indicated that the lunasin peptide had been digested andrendered inactive, thus failing to impact levels of histone H3acetylation.

There was significant reduction in H3 acetylation in the presence ofsynthetic lunasin compared to the untreated control. Both the LeSC andthe LeSC+SF were able to significantly reduce H3 acetylation by PCAF,indicating that the lunasin found in both soy protein extracts isbiologically active. Pancreatin digestion of LeSC+SF reduced thebiological activity but not to the extent observed when LeSC alone isdigested. Like LeSC, soy protein isolate and soy concentrate thatcontain significant amounts of lunasin, did not show lunasin biologicalactivity after pancreatin digestion These results indicate that theformulated LeSC+SF protects lunasin to a certain degree from pancreatindigestion, and allows lunasin to retain its biological activity.

Example 6

Partial digestion of formulated LeSC+SF increases biological activity oflunasin. A confirmatory experiment to determine the biological activityof digested and undigested LeSC and LeSC+SF was conducted using adifferent core histone template. This time we used the core histonesextracted from HeLa tumor cells. Unlike the chicken erythrocyte cells,core histones from sodium butyrate treated HeLa cells are commerciallyavailable (Upstate/Millipore, Piscataway, N.J.), and can be used as apositive control for histone acetylation. The core histones isolatedfrom untreated HeLa cells were used as a negative control (low levels ofhistone acetylation) and as template for the HAT assay.

The HAT bioactivity assay was conducted using acid extracted corehistones from HeLa cells (Upstate/Millipore) as a template (temp (−)control) for the PCAF catalyzed HAT reaction. Core histones from sodiumbutyrate (NaB) treated HeLa cells were used as a positive control sinceNaB is a histone deacetylase inhibitor known to increase histoneacetylation. The inhibitory effect of synthetic lunasin (+synL) onhistone H3 acetylation by PCAF was used to compare the effect oflunasin-enriched soy concentrate (A), digested LeSC (A dig), LeSC+SF (B)and digested LeSC+SF (B dig). LeSC and LeSC+SF were partially digestedby adding pancreatin at 1:0.5 (w/w) and incubating at 38° C. for 15 min.The numbers below the legend indicate relative densitometer readingsnormalized using immunosignal from the template (temp). Low numbersindicate presence of lunasin biological activity.

The results showed that significant reduction in H3 acetylation in thepresence of synthetic lunasin was seen. The undigested LeSC (A) andLeSC+SF (B) showed reduced levels of H3 acetylation, indicating that thenatural lunasin found in these soy extracts was biologically active.Partial digestion of LeSC (A Dig) led to the loss of biologicalactivity.

Surprisingly, partial digestion of LeSC+SF resulted in an increase inbiological activity rather than a decrease. While it is not intendedthat the present invention be limited to any precise mechanism, it isbelieved that lunasin is covalently bound to high molecular weightprotein complexes and that, with the protection of soy flour, partialdigestion only breaks down these bonds and releases, but does notdestroy, bioactive lunasin into the solution. In a preferred embodimentof the present invention, lunasin is partially digested prior to use. Inanother preferred embodiment of the present invention, soy flour ispresent when lunasin is partially digested.

LeSC+SF was partially digested by mixing it with freshly preparedpancreatin solution (10 μg/mL of distilled water) in a 1:0.5, (w/w)ratio. Mixture was incubated at 38° C. for 15 min. before proteases anddigestive enzymes were inactivated by boiling for 5 min and thenquenching in ice. Under these digestion conditions the lunasin in theLeSC soy extract was digested and inactivated while that of LeSC+SF weremore biologically active. However, the conditions for the partialdigestion of LeSC+SF has to be determined empirically by analyzingdigestion products for lunasin content and biological activity using theHAT assay.

Variations in the sources of pancreatin and protease enzymes, the age ofthe protease enzyme, or incubation conditions can lead to variability indigestion conditions. For example, the use of one month old preparationsof pancreatin for partial digestion led to the degradation and loss ofactivity of lunasin under similar incubation conditions described above.Therefore, in a preferred embodiment of the present invention,acceptable ranges for concentration of and incubation time with theprotease enzymes are determined using an assay such as the HAT assayused above to evaluate biological activity of the treated compositions.

Example 7

Chymotrypsin inhibitors (Chy) protect the bioactivity of lunasin. Todetermine which protease inhibitors found in soy protects lunasin fromdigestion, soybean trypsin inhibitor and trypsin+chymotrypsin inhibitorswere obtained from Sigma and mixed with LeSC on 1:1 w/w ratio. Themixtures were digested with pancreatin, and digestion productsimmunostained with lunasin antibody.

Details of the experiment are as follows. LeSC+soybean trypsininhibitors (1:1 w/w) (Sigma) and LeSC+trypsin and chymotrypsininhibitors (1:1 w/w) (Sigma) were digested with pancreatin (1:1 w/w) byincubating at 38° C. for 15 min. Digestion products and LeSC wereanalyzed by Western blot analysis using lunasin primary antibody andsynthetic lunasin as standard controls.

HAT bioactivity assay was conducted using core histones from chickenerythrocyte cells (Upstate/Millipore) as a template for the PCAFcatalyzed HAT reaction. The inhibitory effect of synthetic lunasin(+synL) on histone H3 acetylation by PCAF as compared to the negativeuntreated control (−synL) was used to compare the effect of digestedLeSC (A), digested LeSC+try+chy (B), digested LeSC+try (C), undigestedLeSC (D) and undigested LeSC+SF (E.)

The results showed that in the LeSC+trypsin+chymotrypsin inhibitorssample lunasin was better protected from digestion than in theLeSC+trypsin inhibitor sample. Likewise, in HAT assays to determinelunasin biological activity, digestion of LeSC+trypsin+chymotrypsininhibitors was significantly more bioactive than LeSC+trypsin inhibitor.Pancreatin digestion of LeSC led to the loss of biological activity.These results indicate that the presence of chymotrypsin inhibitors inlunasin-enriched soy concentrate (LeSC) both helps protect thebiological activity of lunasin and helps protect lunasin from excessivedigestion.

Example 8 Screening Assay to Determine Lunasin Biological Activity

Core histones purified from chicken erythrocyte cells were used astemplates in histone acetylase (HAT) reactions using PCAF histoneacetylase enzyme, in the presence or absence of around 2-10 uM lunasin.The core histone template and lunasin-enriched soy concentrates (LeSCand LeSC+SF) were mixed (10:1 w/w) and incubated in ice for 5 min and25° C. for 10 min before mixture was added to 1×HAT reaction mix, 1 uMacetyl CoA and 5 uL PCAF (based on recommended concentration fromUpstate/Millipore). Reaction mixture was incubated at 30° C. whileshaking at 250 rpm for 1 h. Reaction was stopped by adding Laemmli stopbuffer (1:1 v/v) with beta-mercaptoethanol, and boiling for 5 min.before quenching in ice for 15 min. The products of PCAF HAT reactionwere run on 16% SDS-PAGE, blotted onto nitrocellulose membrane andimmunostained with primary antibodies raised against diacetylatedhistone H3 (Ac-Lys 13+Ac-Lys14H3) followed by HRP-conjugated anti-rabbitsecondary antibody. Chemiluminescent signals from antibody complexeswere visualized using standard chemiluminescent reagents and exposed toKodak BioMAX film, developed and spot densitometer measured by usingdigital scanner and UN-SCAN-IT software program from Silk Scientific(Orem, Utah).

The results showed the reduction of H3 acetylation in the reactionmixtures treated with LeSC and LeSC+SF as compared to the untreatedcontrol, indicating that this screening procedure can determine thebiological activity of lunasin-enriched soy concentrates. It was alsodetermined that digestion of LeSC eliminates biological activity but notthat of LeSC+SF which shows only a partial reduction of biologicalactivity.

Example 9

The in vivo activity of the presently described compositions, as well astreatment utilization of kits and treatment methods, may be optionallydetermined by either of the following procedures.

Male dogs (beagles, ranging from about 9 to about 14 kilograms, 1 to 4years old) are fed a standard dog feed supplemented with 5.5% lard and1% cholesterol. Baseline blood samples are drawn from fasted dogs priorto initiating the study to obtain reference values for plasmacholesterol. Dogs are then randomized to groups of five animals withsimilar plasma cholesterol levels. The animals are dosed in accordancewith a treatment method described herein immediately prior to dietpresentation for seven days. Blood samples are obtained 24 hours afterthe last dose for plasma cholesterol determinations. Plasma cholesterollevels are determined by a modification of the cholesterol oxidasemethod using a commercially available kit.

In an optional alternative procedure, hamsters are separated into groupsof six and given a controlled cholesterol diet containing 0.5%cholesterol for seven days. Diet consumption is monitored to determinedietary cholesterol exposure. The animals are dosed in accordance with atreatment method described herein once daily beginning with theinitiation of diet. Dosing is by oral gavage. All animals moribund or inpoor physical condition are euthanized. After seven days, the animalsare anesthetized by intramuscular (IM) injection of ketamine andsacrificed by decapitation. Blood is collected into vacutainer tubescontaining EDTA for plasma lipid analysis and the liver is excised fortissue lipid analysis. Lipid analysis is conducted as per publishedprocedures (e.g., Schnitzer-Polokoff et al., Comp. Biochem. Physiol.,99A, 4 (1991), pp. 665-670 and data is recorded as percent reduction oflipid versus control.

REFERENCES

The numeric references incorporated above in parentheses correspond tothe following list of published papers and abstracts. All of the belowlisted publications are herein incorporated by reference in theirentirety to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein its entirety.

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1. A method for protecting against photoaging of skin in an individual,comprising: (a) providing: (i) an individual desiring to preventphotoaging of skin and, (ii) a composition comprising a compoundselected from the group consisting of the peptide of SEQ ID NO 2 and afunctionally equivalent variant, fragment or analogue of said peptide;and (b) administering said composition to said subject to protectagainst photoaging.
 2. The method of claim 1, wherein said compound isobtained from soybean, wheat or barley.
 3. The method of claim 1,wherein said compound is obtained by producing, extracting and purifyingsaid compound using recombinant DNA techniques.
 4. The method of claim1, wherein said compound is obtained by synthetic polypeptideproduction.
 5. The method of claim 1, wherein said individual is ahuman.
 6. The method of claim 1 wherein administering comprises topicaladministration of the composition.
 7. The method of claim 1, wherein thecomposition is in the form of a semi-solid formulation, liquid, gel,suspension, or aerosol spray.
 8. The method of claim 1, wherein saidcomposition further comprises chymotrypsin inhibitor.
 9. The method ofclaim 1, wherein said compound is administered to said individual atbetween 5 μg/ml and 50 μg/ml.
 10. A method for protecting againstcollagen breakdown in the skin of an individual, comprising: (a)providing: (i) an individual desiring to prevent collagen breakdown inthe skin and, (ii) a composition comprising a compound selected from thegroup consisting of the peptide of SEQ ID NO 2 and a functionallyequivalent variant, fragment or analogue of said peptide; and (b)administering said composition to said subject to protect againstcollagen breakdown.
 11. The method of claim 10, wherein said compound isobtained from soybean, wheat or barley.
 12. The method of claim 10,wherein said compound is obtained by producing, extracting and purifyingsaid compound using recombinant DNA techniques.
 13. The method of claim10, wherein said compound is obtained by synthetic polypeptideproduction.
 14. The method of claim 10, wherein said individual is ahuman.
 15. The method of claim 10 wherein administering comprisestopical administration of the composition.
 16. The method of claim 10,wherein the composition is in the form of a semi-solid formulation,liquid, gel, suspension, or aerosol spray.
 17. The method of claim 10,wherein said composition further comprises chymotrypsin inhibitor. 18.The method of claim 10, wherein said compound is administered to saidindividual at between 5 μg/ml and 50 μg/ml.
 19. A method for protectingagainst wrinkling of the skin in an individual, comprising: (a)providing: (i) an individual desiring to prevent wrinkling of skin and,(ii) a composition comprising a compound selected from the groupconsisting of the peptide of SEQ ID NO 2 and a functionally equivalentvariant, fragment or analogue of said peptide; and (b) administeringsaid composition to said subject to protect against wrinkling of theskin.
 20. The method of claim 19, wherein said compound is obtained fromsoybean, wheat or barley.
 21. The method of claim 19, wherein saidcompound is obtained by producing, extracting and purifying saidcompound using recombinant DNA techniques.
 22. The method of claim 19,wherein said compound is obtained by synthetic polypeptide production.23. The method of claim 19, wherein said individual is a human.
 24. Themethod of claim 19 wherein administering comprises topicaladministration of the composition.
 25. The method of claim 19, whereinthe composition is in the form of a semi-solid formulation, liquid, gel,suspension, or aerosol spray.
 26. The method of claim 19, wherein saidcomposition further comprises chymotrypsin inhibitor.
 27. The method ofclaim 19, wherein said compound is administered to said individual atbetween 5 μg/ml and 50 μg/ml.